Cloning characterization of iaaM, a virulence determinant of Pseudomonas savastanoi

Abstract

Genes for indoleacetic acid production (iaaM and iaaH) are necessary for gall induction by the olive pathogen Pseudomonas savastanoi. In strain 2009 these determinants are borne on plasmid pIAA1. To map and characterize the genes, fragments of pIAA1 generated by EcoRI endonuclease treatment were cloned in Escherichia coli by using plasmid RSF1010 as vector. We isolated a recombinant plasmid encoding iaaM, the locus for tryptophan 2-monooxygenase. This plasmid, called pLUC1, was characterized by restriction endonuclease hydrolysis. It contained a 2.75-kilobase-pair segment of pIAA1. By cloning this segment in the EcoRI site of pBR328 and pBRH3B we showed that efficient expression of iaaM was dependent on the orientation with respect to the vector promoters, and thus determined the direction of transcription. To more finely map iaaM and confirm the orientation of transcription, plasmid pLUC1 was subjected to transposon Tn/mutagenesis. The promoter-distal end of iaaM was mapped between coordinates at 1.7 and 2.15 kilobase pairs of the cloned segment.